ÃÞ»¨ÌÇÖ±²¥

The majority of materials we rely on in our built environment come from chemicals derived from petroleum (petrochemicals), which is a limited resource. One strategy to address concerns surrounding this dwindling resource is to shift away from petrochemicals and toward renewable resources and chemicals produced by living organisms. This framework is termed the "bioeconomy." An emerging international interest in the bioeconomy is helping drive research aimed at providing tangible answers to questions of sustainability, and microorganisms possess many of the metabolic tools needed for this endeavor to be successful.

Closely related to the idea of the bioeconomy is the concept of circularity. Circular products and materials are truly recyclable, rather than degraded as in chemical (traditional) recycling. Once again, microbes are uniquely suited to carry out metabolic processes that breakdown complex products and rebuild them into usable resources. Yet, many of the microorganisms that might drive the bioeconomy do not naturally possess the genetic tools that would enable their industrialization, and researchers are utilizing advances in microbial engineering to circumvent these challenges. 

Developing Organisms for the Bioeconomy

Often, the entire metabolic pathway required for circularity may not be found in nature. Rather, different steps in the breakdown and recycling of complex materials might be found in more than one organism. This distribution of function between multiple microorganisms can be inconvenient in an industrial setting, as each organism will have individual growth requirements that must be met, adding complexity to the process. One approach to address the complexity of community maintenance is to combine all of the necessary genes into 1 organism for biomanufacturing. This approach is termed Consolidated Bioprocessing (CBP). CDP aims to streamline complex community function by packaging the entire breakdown and rebuilding of chemicals of interest into a single organism. 

CBP can be applied in many contexts. For example, utilizing biological processes to produce aviation fuel from plant biomass would require a CBP organism. This organism would break down complex plant biomass and chemically convert it into the desired final product. The goal of implementing CBP would be to simplify processing and reduce costs to make industrialization realistic. 

Yet, producing a CBP organism can require significant genetic engineering if there is not a single, naturally occurring organism that contains all the necessary genes to breakdown a given material and build it into a usable resource. Genetic engineering encompasses a range of techniques that are used to alter an organism's existing genes to produce certain phenotypes, including performing a function of interest. Often, the goal is to add new genes that confer a desired trait to the target organism. The organism chosen as the starting point for engineering is often called the chassis organism. But how do researchers and industrial microbiologists choose the ideal chassis organism for their processes?

One might be inclined to choose a model organism like Escherichia coli or Bacillus subtilis for which well-established genetic protocols already exist, but it turns out that unique engineering questions require unique strategies, which often direct scientists' attention to . For example, does the process of interest occur under challenging conditions, like high temperatures, or produce byproducts that are toxic to some organisms? In those instances, it is important for scientists to choose a thermophilic organism, or an organism that is resistant to toxic byproducts, respectively. However, very few of these traits are present in model organisms, so it might instead be simpler to develop tools to genetically manipulate non-model organisms.

Genetic Engineering Strategies

DNA Uptake: Electroporation, Conjugation and Native Competence

Working with non-model organisms, like and can present significant . First, scientists must encourage the microbe of interest to "take up" or incorporate foreign genetic material into its genome. Typical methods that scientists use to encourage organisms in the laboratory to take up DNA that is not native to their own genomes include electroporation, conjugation and native competence.

• Electroporation: This technique uses an electric shock to create temporary pores in the cell envelope, which then allow DNA to pass into the cell. Electroporation is commonly used technique, and there isn’t much innovation in this space. However, improvements in high throughput approaches, like liquid-handling robotics, offer an exciting way to increase the rate of screening various conditions (e.g., different electroporation media and voltages for electroporation) that might enable DNA uptake.
• Conjugation: This technique uses a donor bacteria to transfer DNA to the bacteria of interest via a pilus upon contact.
• Native competence: This technique relies on a phenotype that some organisms possess (called native competence), which allows cells to uptake DNA from the environment and incorporate it into their genomes. Importantly, not all species possess this ability, and the conditions that cause competence are strain specific.

DNA Maintenance: Restriction Modification Systems and Marker Selection 

After getting the DNA into the cell, it is important to be able to maintain it. In other words, target DNA must be replicated, transcribed and translated as part of the organism's normal biological processes. DNA maintenance and subsequent gene expression in a bacterial host can be hampered by bacterial defense systems. For example, restriction modification systems enable a bacterium to identify ‘self’ DNA by scanning intracellular DNA with restriction enzymes for specific methylation patterns, then degrading DNA that is not methylated properly. The pattern of methylation on a microorganism's genome is called the methylome. Most DNA that scientists try to transform into bacteria of interest are either unmethylated or methylated according to a cloning strain of E. coli’s methylation pattern. Thus, if the DNA scientists are trying to introduce is determined to be non-self, restriction modification systems will subsequently degrade it.

Characterizing the restriction modification systems of the is a technique that is becoming increasingly popular, helping to circumvent natural bacterial defenses and encourage the microbe of interest to accept the newly introduced DNA into its genome. This is accomplished by sequencing the methylome of the microbe using techniques like Oxford Nanopore’s Minion sequencing or with Pacbio’s HIFI sequencing. After computationally identifying methylation patterns, researchers can identify the proteins that produce that methylation pattern, methylases. Once the methylase is identified, it can be heterologously expressed in a strain of E. coli, which will then methylate DNA, such as a plasmid, to match the methylation pattern of the organism of interest, so it is less likely to be degraded once transformed. This ability to mimic methylation patterns is rapidly increasing the plausibility and frequency of genetically engineering new chassis organisms.

Another hurdle for DNA maintenance and long term utility of the organism is marker selection. Many genome editing strategies utilize a selectable marker, such as an antibiotic resistance gene, to selectively grow organisms that have been edited. At industrial scales, the cost of growth media is even more important than in standard laboratory scale experiments. Thus, it is useful to avoid plasmid-based gene expression systems, which often require the addition of antibiotics to growth media for maintenance.

In the absence of plasmid-based expression systems, genes must be integrated into the host’s genome, which can be challenging if the organism does not have highly active homologous recombination machinery. One methodology that has been leveraged to overcome this issue is called Serine-Integrase Assisted Genome Engineering ( In this process, genetic cargo (a set of genes), is flanked on either side by specific DNA that a recombination protein recognizes and uses to integrate into specific DNA sequences in the genome. Compared to more familiar tyrosine recombinases, like Flp and Cre, serine recombinases use 2 unique sites, rather than highly similar sites that are subject to later recombination and potential instability. SAGE’s ability to be used in many microorganisms makes it a promising tool for non-model microbial chassis.

While this article highlights only a few genetic strategies to expand genetic technology to new microorganisms, many may be on the horizon. Expanding our genetic repertoire is an important step in realizing a microbially manufactured future. To achieve this, we need to develop highly efficient techniques for genome modification in any organism. Microbes have long been our partners, and the more we come to understand about them, the more we can leverage them to perform useful tasks, like replacing petroleum-based plastics and fuels with biobased biodegradable plastics and biofuels.

---

Microbes have long greased the gears of the bioeconomy. The Spring 2024 issue of Microcosm, our flagship member magazine, takes an in-depth look at the critical role of microbes in creating sustainable future for all.